García M C
Departamento de Química Analítica, Facultad de Química, Universidad de Alcalá, 28871 Alcalá de Henares, Madrid, Spain.
J Chromatogr B Analyt Technol Biomed Life Sci. 2005 Oct 25;825(2):111-23. doi: 10.1016/j.jchromb.2005.03.041. Epub 2005 Apr 26.
The study of the effect of mobile phases on sensitivity in the analysis of peptides and proteins by high-performance liquid chromatography (HPLC)-electrospray mass spectrometry (ESI-MS) has been the aim of this review. Reversed-phase chromatography (RPLC) is the chromatographic mode most suitable for coupling with ESI-MS since mobile phases containing organic modifiers are used. The analysis of proteins and peptides by RPLC mostly involves the use of trifluoroacetic acid (TFA) as an ion-pairing agent despite its being a strong suppressor of the MS signal. Different studies reporting the effects of using other ion-pairing agents (other perfluorinated acids, acetic acid, formic acid, etc.) and buffers (ammonium acetate, ammonium formate, ammonium bicarbonate, morpholine, etc.) in RPLC-ESI-MS of proteins and peptides did not yield a single strong candidate that could generally replace TFA. The enhancement in sensitivity with other reagents observed in some cases strongly depended on the analyte, the experimental conditions used, and the mass spectrometer and, usually, it did not compensate for the loss in separation resolution related to TFA. The examples of direct coupling of affinity, size-exclusion, or ion-exchange chromatography (IEC) to ESI-MS are very limited because of incompatibilities related to the use of mobile phases containing high salt concentrations. To overcome this problem, an intermediate desalting step is needed. Multidimensional chromatography, microdialysis, and ion-capture modules can be used to couple these chromatographic modes with ESI-MS. Multidimensional chromatography with RPLC as a second dimension has most often been used. Although most examples involve the trap and analysis in the second dimension of a certain part of the first separation, some comprehensive analyses of the entire sample in the second dimension have also appeared.
本综述旨在研究高效液相色谱（HPLC）-电喷雾质谱（ESI-MS）分析肽和蛋白质时流动相对灵敏度的影响。反相色谱（RPLC）是最适合与ESI-MS联用的色谱模式，因为使用了含有有机改性剂的流动相。尽管三氟乙酸（TFA）是质谱信号的强抑制剂，但RPLC分析蛋白质和肽大多使用TFA作为离子对试剂。不同研究报道了在蛋白质和肽的RPLC-ESI-MS中使用其他离子对试剂（其他全氟酸、乙酸、甲酸等）和缓冲液（乙酸铵、甲酸铵、碳酸氢铵、吗啉等）的效果，但没有产生一个能普遍替代TFA的有力候选物。在某些情况下观察到的其他试剂灵敏度的提高很大程度上取决于分析物、所用的实验条件和质谱仪，而且通常无法弥补与TFA相关的分离分辨率损失。由于与使用高盐浓度流动相相关的不相容性，亲和色谱、尺寸排阻色谱或离子交换色谱（IEC）与ESI-MS直接联用的例子非常有限。为克服这一问题，需要一个中间脱盐步骤。多维色谱、微透析和离子捕获模块可用于将这些色谱模式与ESI-MS联用。最常使用以RPLC作为第二维的多维色谱。虽然大多数例子涉及在第二维中对第一维分离的某一部分进行捕集和分析，但也出现了一些在第二维中对整个样品进行全面分析的情况。
